Benzodiazepines are widely used to treat anxiety, insomnia and seizure disorders. Furthermore they are key adjunct treatments in schizophrenia, depression and alcohol detoxification. However, the use of these safe and efficacious compounds is severely limited due to the development of tolerance. Benzodiazepines potentiate the activity of 3-aminobutyric acid (GABAA) receptors, the major inhibitory neurotransmitter receptors in the central nervous system. The molecular mechanism underlying the development of tolerance has not yet been determined. It is increasingly evident that benzodiazepines preferentially enhance the activity of GABAA receptor subtypes present at synaptic sites that are largely composed of ?1-3, ? and ?2 subunits. In contrast, the majority of extrasynaptic GABAA receptors (GABAARs), which mediate tonic inhibition, have structures different from those of their synaptic counterparts and are insensitive to functional modulation by benzodiazepines. However, a mechanism linking benzodiazepine treatment of neurons to changes in surface levels of GABAAR subtypes and the efficacy of neuronal inhibition has not been demonstrated. These phenomena together with our preliminary studies generated the central hypothesis driving the experiments described in this proposal: Exposure of neurons to benzodiazepines promotes the removal of ?2 subunit-containing GABAARs from the plasma membrane and their subsequent degradation, leading to a reduction in inhibitory synapse size and number along with a decrease in the efficacy of synaptic inhibition. Our proposal centers on three specific aims: (1) We will characterize the effects of benzodiazepine (BZ) treatment on GABAAR membrane trafficking and degradation;(2) We will measure the effects of BZ treatment on synaptic inhibition and identify the mechanism altering synaptic efficacy;and (3) We will measure the ability of BZ treatment to modulate cell surface accumulation of GABAARs and synaptic inhibition in wild type mice and ?2H101R mutant mice that express ?2-containing GABAAR that are BZ insensitive. PUBLIC HEALTH RELEVANCE: Benzodiazepines are widely used to treat anxiety, insomnia and seizure disorders, but their clinical use is severely limited due to the development of tolerance. The therapeutic actions of benzodiazepines are primarily exerted by potentiating the activity of ?-aminobutyric acid (GABA) type A receptors, the major inhibitory neurotransmitter receptors in the central nervous system. We will investigate the effects of benzodiazepines on GABA type A receptors to define a molecular mechanism underlying benzodiazepine tolerance, facilitating the development of new therapeutic agents to improve patient quality of life.